Background/Objective: Development of a new enzyme-linked immunosorbent assay (ELISA) for screening human blood serum and plasma for antibodies to human immunodeficiency virus type 1 (HIV1) and type 2 (HIV2) as HIV1/2REC ELISA diagnostic kit based on E. coli derived soluble recombinant proteins. Methods: Polypeptides corresponding to HIV1 gp41 and HIV2 gp36 immunodominant regions and HIV1 gag were expressed in E. coli in fusion with thioredoxin (Trx) to obtain a highly purified (>98%) soluble refolded proteins, which was used as solid phase antigens for ELISA. Results: The sensitivity and specificity of anti-HIV1/2 antibody detection were evaluated with representative panels of positive and negative sera. Positive panels included HIV1-positive Western-blot (WB)-confirmed specimens collected in Iran, Russia, and Uganda. Commercially available HIV1 and HIV2 seroconversion low titer and performance panels were also used. Negative panel was collected from random volunteer blood donors, risk group members, HCV-infected patients and individuals with non-HIV related conditions potentially influencing test results. The sensitivity of antibody detection with new kit was determined to be 100%. Specificity was determined to be 99.82%. It was shown than thioredoxin (Trx) did not change the immunodominant epitopes of HIV. These fusion proteins are recognized by human native antibodies. In addition, thioredoxin (Trx) would help natural refolding of HIV proteins by E. coli. Conclusion: These characteristics of the new assay are comparable to those of majority of FDA-licensed and officially approved European diagnostic kits, which are currently available in the United States and Europe.